Both animals and plants use steroids as signaling molecules (hormones) for physiological and developmental regulation. In plants, brassinosteroids (BRs) have growth-promoting activity and are essential for normal growth and development. Deficiencies in BR biosynthesis and responsiveness cause dramatic developmental alterations that include dwarfism due to reduced cell elongation and expansion, male sterility, delayed reproductive development, and development of light-grown morphology in the dark. BRs are perceived by the cell surface receptor kinase BAIl. How the BR signal is transduced from BRI1 on the plasma membrane to other cellular and nuclear responses remains unknown. This proposal aims at obtaining a molecular understanding of the BR signaling pathways in the model plant Arabidopsis thaliana, using approaches of molecular genetics, biochemistry, and cell biology. First, we have found that the BRI1 receptor kinase is in a large protein complex, and we hypothesize that other proteins associated with BRI1 in the receptor complex are required for the receptor function. We plan to identify the proteins associated with BRI1 using affinity purification and to characterize the functions of these proteins using reverse-genetic and molecular approaches. Second, we have isolated and carried out preliminary studies of a new BR-signaling mutant bzr1-1 D and the corresponding gene BZR1. Based on these studies, we hypothesize that BZR1 functions as a positive regulator in the BR response pathway downstream of BRI1. We plan to further characterize the BZR1 gene by identifying loss-of-function alleles of bzrl and bzr2, by analyzing the interaction of BZR1 with other genes of the BR signaling pathway, and by determining the biochemical activity of the BZR1 protein. We also plan to identify and characterize BZR1 interacting proteins and perform genetic studies of intragenic and extragenic mutations that suppress or enhance the bzr1 -1 D mutant phenotype. Finally, we have screened and isolated additional mutants associated with BR signaling. We have cloned the gene corresponding to one of these mutants, BZR6, which encodes a small protein. We hypothesize that BZR6 may function as a peptide ligand antagonizing BR signaling or as one of the secondary signals for the BR response. We plan to determine the functions of BZR6 and its homologue BZL1.